The site of RNA processing and the spatial relationship between RNA processing and transcription in mammalian nuclei is a subject of a long-standing interest in cell biology. There is evidence for and against the cotranscriptional splicing of precursor mRNAs. A. L. Beyer and Y. N. Osheim (Semm. Cell Biol. 2:131 (1991)) have shown that nascent transcripts in Drosophila are associated with spliceosomes, and in some cases, the spliceosome and nascent transcripts can be directly visualized by electron microscopy. In contrast, J. R. Nevins (Annu. Rev. Biochem. 52:441 (1983)) and others have shown that the unspliced transcripts in mammalian cells can be isolated in the poly(A) fraction, indicating that splicing is posttranscriptional.
Prior art studies on whether or not mRNA transcripts in mammalian cells are localized at the site of transcription or are free to diffuse within the nucleus after synthesis have yielded inconsistent results. Those studies that have reported localized foci of RNAs have failed to establish the significance of such localizations. Highly localized nuclear "tracks" of specific viral mRNAs have been observed by fluorescence microscopy in chromatin-depleted nuclear matrix extracts (J. B. Lawrence, R. H. Singer and L. M. Marselle. Cell 57:493 (1989); Y. Xing and J. B. Lawrence. J. Cell Biol. 112:1055 (1991)). Several viral RNAs have been observed to localize at only one or two sites within the nucleus (J. B. Lawrence, et al. (1989). supra; J. B. Lawrence, L. M. Marselle, K. S. Byron, J. L. Sullivan and R. H. Singer. Proc. Natl. Acad. Sci. USA 87: 5420 (1990); A. Raap et al. Exp. Cell Res. 197:319 (1991)), but this observation is in contrast to microinjected globin RNA, which appears to diffuse within the nucleus. Lawrence, et al. (1990) have speculated that these tracks might represent sites of transcription, but have not ruled out other functions e.g., sites of RNA processing, RNA transport, or some other cellular function. Moreover, they did not spatially correlate the foci with any particular active genes.
Total nuclear polyadenylate RNA (poly(A) RNA) has been shown to accumulate in 20 to 50 discrete "transcript domains" which coincide with the location of small nuclear ribonucleoproteins (snRNPS) (K. C. Carter, K. L. Taneja and J. B. Lawrence. J. Cell Biol. 115:1191 (1991)). These snRNPs have previously been reported to exhibit a clustered nuclear distribution coincident with the spliceosome assembly factor SC-35. Although the association of poly(A) RNA with snRNPs in these studies was observed using hybridization of probes to DNA and mRNA in the same cell, it was not possible to make any conclusions concerning spatial correlation of transcription and processing of a specific RNA with expression of a specific gene because the DNA-specific probe was directed to untranscribed centromere DNA and the mRNA-specific probe oligo(dT) hybridized to total mRNA.